ARDS is a major cause of morbidity and mortality in the United States. There are no specific therapies for ARDS other than protective mechanical ventilation and conservative fluid management and mortality remains high at 30-50%. Thus, there is a critical need for specific therapies that target fundamental mechanisms of ARDS. Increased lung epithelial permeability is a pathophysiologic hallmark of ARDS and is manifest clinically by pulmonary edema, impaired gas exchange and acute respiratory failure. We have identified cell free hemoglobin as a novel mediator of increased epithelial permeability in ARDS. Our preliminary data show that airspace levels of cell-free hemoglobin are high in ARDS and are associated with increased lung permeability and poor clinical outcomes. Furthermore, it is the Ferryl oxidized form of hemoglobin that is elevated in the airspaces in ARDS. In a clinical trial in severe sepsis, the leading cause of ARDS, treatment with an inhibitor of Ferryl hemoglobin, acetaminophen, decreased lipid peroxidation and attenuated acute kidney injury. In cultured lung epithelial cells, hemoglobin treatment induces epithelial barrier permeability through cellular and mitochondrial reactive oxygen species generation. Furthermore, intratracheal administration of cell-free hemoglobin to the lungs of mice leads to generation of oxidized Ferryl hemoglobin and increased lung alveolar capillary barrier permeability. These findings support the overall hypothesis for the proposed studies, that formation of Ferryl-Hgb in the airspace in ARDS causes alveolar epithelial cell oxidative stress and increased epithelial permeability, contributing to the pathophysiology of ARDS. To test this hypothesis I have assembled a cross-disciplinary team of co-Investigators and consultants with expertise in clinical ARDS (Ware), Hgb redox chemistry (Roberts), primary alveolar epithelial cell isolation and culture (Guttentag), mitochondrial oxidative stress (Dikalov), human models of ARDS (Matthay) and advanced in vivo imaging techniques (West) whose expertise complements my own in cellular models of epithelial permeability and animal models of ARDS. We will utilize clinical samples of pulmonary edema fluid already collected from patients with ARDS and hydrostatic pulmonary edema along with mouse and epithelial cell culture studies to show that levels of Ferryl hemoglobin in the airspace are increased in human ARDS and cause increased lung epithelial permeability in mice and cultured epithelial cells (Aim 1). We will define the cellular and molecular mechanism of hemoglobin induced increases in epithelial permeability and cellular and mitochondrial oxidative stress in the epithelium (Aim 2). Finally we will test a therapy targeted at Ferryl hemoglobin (acetaminophen) in cell culture, in a clinically relevant model of ventilator induced lung injury in mice and in an isolated perfused human lung model (Aim 3). This proposal is highly innovative and addresses a clinically significant problem. Results from these studies will greatly advance our understanding of the mechanisms of lung epithelial permeability in acute lung injury and will pave the way for future novel targeted therapeutics in ARDS.